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Key Specifications Table
| Species Reactivity | Key Applications |
|---|---|
| H, M | RIP, WB, IP |
| Description | |
|---|---|
| Catalogue Number | 03-196 |
| Brand Family | Upstate |
| Trade Name |
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| Description | RIPAb+™ EED - RIP Validated Antibody and Primer Set |
| Overview | RIPAb+ antibodies are evaluated using the RNA Binding Protein Immunoprecipitation (RIP) assay. Each RIPAb+ antibody set includes a negative control antibody to ensure specificity of the RIP reaction and is verified for the co-immunoprecipitation of RNA associated specifically with the immunoprecipitated RNA binding protein of interest. Where appropriate, the RIPAb+ set also includes quantitative RT-PCR control primers (RIP Primers) to biologically validate your IP results by successfully co-precipitating the specific RNA targets, such as messenger RNAs. The qPCR protocol and primer sequences are provided, allowing researchers to validate RIP protocols when using the antibody in their experimental context. If a target specific assay is not provided, the RIPAb+ kit is validated using an automated microfluidics-based assay by enrichment of detectable RNA over control immunoprecipitation. Transcriptional repression mediated by EED involves histone deacetylation. EED protein is present in four isoforms. These EED isoforms selectively associate with distinct EZH2-containing complexes, resulting in differential targeting of their associated methyltransferase activity. |
| Alternate Names |
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| Background Information | Polycomb group proteins are important for maintaining transcriptional silencing. One conserved PcG complex, PRC2, is composed of several proteins including the histone methyltransferase EZH2, the WD-repeat protein EED (Embryonic ectoderm development), and the Zn-finger protein Suz12. Transcriptional repression mediated by EED involves histone deacetylation, while the EZH2 methylates histone H3 on lysine 27. EED protein is present in four isoforms. These EED isoforms selectively associate with distinct EZH2-containing complexes, resulting in differential targeting of their associated methyltransferase activity. These complexes play a role in Hox gene silencing, X-inactivation, germline development, stem cell pluripotency and cancer metastasis. |
| Product Information | |
|---|---|
| Format | Purified |
| Control |
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| Presentation | Anti-EED (Mouse Monoclonal). One vial containing 50 μg of protein G purified antibody in 65 μL of 0.1 M Tris-Glycine (pH7.4) 150 mM NaCl, containing 0.05% azide before the addition of 30% glycerol. Store at -20°C . Normal Mouse IgG. One vial containing 125 µg of purified mouse IgG in 125 µL of storage buffer containing 0.1% sodium azide. Store at -20°C. RIP Primers, lincSFPQ. One vial containing 75 μL of 5 μM of each primer specific for the human lincRNA SFPQ. Store at -20°C. FOR: TCG TAC TGT TAG GCC CTT GG REV: AAC CTT GCA TGA AGA GCA CC |
| Quality Level | MQ100 |
| Applications | |
|---|---|
| Application | This RIPAb+ EED -RIP Validated Antibody & Primer Set conveniently includes the antibody & the specific control PCR primers. |
| Key Applications |
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| Application Notes | Immunoprecipitation from RIP lysate: Representative lot data. RIP lysate from HeLa cells (~2 X 10E7 cell equivalents per IP) was subjected to immunoprecipitation using 5 µg of either a normal mouse IgG, (Cat. #CS200621), or 5 µg of Anti-EED antibody (Cat. # CS204372). Ten percent of the precipitated proteins (lane 1: mouse IgG, lane 3: EED) and HeLa whole cell lysate (lane 2) were resolved by electrophoresis, transferred to nitrocellulose and probed with Anti-EED antibody (Cat. # CS204372, 1:5000). Proteins were visualized using One-Step™ IP-Western kit (GenScript Cat. # L00232). Arrow indicates EED. Western Blot Analysis: Representative lot data. A431 cell lysate was resolved by electrophoresis, transferred to nitrocellulose and probed with Anti-EED (1:5,000 dilution). Proteins were visualized using a goat anti-mouse IgG conjugated to HRP and a chemiluminescence detection system. Arrow indicates EED (~50 kDa). |
| Biological Information | |
|---|---|
| Immunogen | Recombinant fusion protein of full length murine EED tagged with MBP. |
| Concentration | Please refer to the Certificate of Analysis for the lot-specific concentration. |
| Host | Mouse |
| Specificity | This antibody recognizes mouse EED, (~30-65 kDa). |
| Isotype | IgG2aκ |
| Species Reactivity |
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| Species Reactivity Note | Human, Mouse. Not tested in other species. |
| Antibody Type | Monoclonal Antibody |
| Entrez Gene Number |
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| Gene Symbol |
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| Purification Method | Protein G Purified |
| UniProt Number |
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| UniProt Summary | FUNCTION: Polycomb group (PcG) protein. Component of the PRC2/EED-EZH2 complex, which methylates Lys-9 and Lys-27 of histone H3, leading to transcriptional repression of the affected target gene. The PRC2/EED-EZH2 complex may also serve as a recruiting platform for DNA methyltransferases, thereby linking two epigenetic repression systems. Genes repressed by the PRC2/EED-EZH2 complex include HOXC8, HOXA9, MYT1 and CDKN2A. Ref.2 Ref.9 Ref.14 Ref.16 Ref.17 Ref.19 Ref.22 Ref.26 SUBUNIT: Interacts with MLL By similarity. Component of the PRC2/EED-EZH2 complex, which includes EED, EZH2, SUZ12, RBBP4 and RBBP7 and possibly AEBP2. The minimum components required for methyltransferase activity of the PRC2/EED-EZH2 complex are EED, EZH2 and SUZ12. The PRC2 complex may also interact with DNMT1, DNMT3A, DNMT3B and PHF1 via the EZH2 subunit and with SIRT1 via the SUZ12 subunit. Interacts with HDAC, HDAC2, histone H1 and YY1. May interact with ITGA4, ITGAE and ITGB7. May interact with the MA protein of HIV-1. SUBCELLULAR LOCATION: Nucleus. Note= Transiently colocalizes with XIST at inactive X chromosomes. Ref.2 Ref.17 Ref.3 Ref.12 Ref.15 SPECIFICITY: Expressed in brain, colon, heart, kidney, liver, lung, muscle, ovary, peripheral blood leukocytes, pancreas, placenta, prostate, spleen, small intestine, testis, thymus and uterus. Appears to be overexpressed in breast and colon cancer. Ref.2 Ref.3 Ref.1 Ref.8 Ref.20 Developmental stage Expression peaks at the G1/S phase boundary. Ref.14 Induction Expression is induced by E2F1, E2F2 and E2F3. Ref.14 SIMILARITY: Belongs to the WD repeat ESC family. Contains 7 WD repeats. Caution Two variants of the PRC2 complex have been described, termed PRC3 and PRC4. Each of the three complexes may include a different complement of EED isoforms, although the precise sequences of the isoforms in each complex have not been determined. The PRC2 and PRC4 complexes may also methylate Lys-26 of histone H1 in addition to Lys-27 of histone H3 (Ref.18 and Ref.20), although other studies have demonstrated no methylation of Lys-26 of histone H1 by PRC2 (Ref.21). |
| Molecular Weight | Ranges from 30-65 kDa (5-6 isoforms of EED are detected). |
| Product Usage Statements | |
|---|---|
| Quality Assurance | RNA Binding Protein Immunoprecipitation: Representative lot data. RIP Lysate prepared from HeLa cells (2 X 10E7 cell equivalents per IP) were subjected to immunoprecipitation using 5 µg of either a normal mouse IgG or 5 µg of Anti-EED antibody and the Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Cat. # 17-700). Successful immunoprecipitation of EED-associated RNA was verified by qPCR using RIP Primers lincSFPQ. Please refer to the Magna RIP™ (Cat. # 17-700) or EZ-Magna RIP™ (Cat. # 17-701) protocol for experimental details. |
| Usage Statement |
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| Storage and Shipping Information | |
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| Storage Conditions | Stable for 1 year at -20°C from date of receipt. Handling Recommendations: Upon receipt, and prior to removing the cap, centrifuge the vial and gently mix the solution. Aliquot into microcentrifuge tubes and store at -20°C. Avoid repeated freeze/thaw cycles, which may damage IgG and affect product performance. Note: Variabillity in freezer temperatures below -20°C may cause glycerol containing solutions to become frozen during storage. |
| Packaging Information | |
|---|---|
| Material Size | 10 assays |
| Material Package | 10 assays per set. Recommended use: ~5 μg of antibody per RIP (dependent upon biological context). |